Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide, affecting over 2 million individuals 45 years of age or older in the US. The personal, social, and medical burden of glaucoma within the VHA remains an extraordinarily significant concern. Despite its overwhelming prevalence and socioeconomic impact, the treatment of Veterans with POAG is currently restricted to non-specific interventions that lower elevated intraocular pressure (IOP). For many glaucomatous Veterans, pharmacological management of IOP remains clinically refractive. These Veterans often require more extensive interventions, including invasive surgical manipulation. The development of targeted therapeutic strategies directed at the cause of elevated IOP is critical for the management of our glaucomatous Veterans. In healthy eyes, IOP is maintained through balanced production and outflow of aqueous humor (AH). Increased resistance to AH outflow through the trabecular meshwork (TM) is considered to be a major contributor of aberrant elevation of IOP in Veterans with POAG. The mechanism by which this occurs remains poorly defined, but most likely involves a combination of enhanced TM cell contractility and altered ECM deposition. Independent studies have shown that TGF-2, a cytokine that facilitates Rho GTPase-mediated ECM deposition and actin stress fiber organization in TM cells, is markedly elevated in the AH of POAG patients. Endothelin-1 (ET-1) a potent vasoconstrictor, is also increased in the AH from POAG patients. The association between TGF-2, ET-1, and Rho GTPase signaling with POAG, however, remains unclear. Peer-reviewed and published studies from our laboratory demonstrate a striking association between TGF-2 and ET-1 production within human TM cells. Our preliminary findings included in this application further demonstrate for the first time that by selectively targeting and disrupting Rho GTPase signaling, we can successfully prevent harmful increases in TGF-2 and ET-1 expression in human TM cells. These findings strongly suggest that aberrant Rho GTPase signaling elevates IOP, in part, by facilitating pathologic increases in TGF-2 and ET-1 in the anterior segment of POAG Veterans. Novel translational studies are critically needed to develop therapeutic and rehabilitative strategies for the advanced care of our Veterans debilitated by glaucoma. siRNA technology is an exciting and promising new field that has realistic therapeutic/rehabilitative potential for the management of ocular disorders, including glaucoma. In this two-year SPiRE study, we will determine whether siRNA-mediated Rho GTPase knockdown therapeutically lowers experimentally-elevated IOP. Hypothesis: Therapeutically administered siRNA selectively targeting monomeric Rho GTPases will markedly lower pathologically elevated intraocular pressure by disrupting TGF-2 and ET-1 expression and signaling. Specific Aim 1 will determine whether administration of siRNAs targeting key monomeric Rho GTPases protects against experimentally elevated changes in outflow resistance as quantified by human and porcine anterior segment perfusion analyses. Specific Aim 2 will determine whether administration of siRNAs targeting key monomeric Rho GTPases attenuates expression of proET-1, ETA, ETB, TGF-2 or TGFRI in human and porcine globes with experimentally elevated outflow resistance as quantified by immunohistochemistry and confocal microscopy. The immediate goal of this study is to establish siRNA-mediated Rho GTPase knockdown as a novel strategy by which to manage elevated IOP associated with POAG. Successful completion of this study will serve as a critical first step toward developing this technology for the management of Veterans with POAG.